Citrus is one of the most important crops and is grown worldwide. Global citrus production in 2013 exceeded 88 million metric tons, with an estimated value of $9 billion. New cultivars with desirable traits have been developed to improve citrus yield and nutritional value, as well as its capacity to adapt to biotic and abiotic stresses. However, conventional breeding is greatly challenged due to many limitations, e.g., narrow genetic diversity and long juvenile period. New technologies can improve citrus for disease resistance against citrus canker, citrus Huanglongbing and other diseases. Among them, citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases and most commercial citrus varieties are susceptible to Xcc.
Presently, three technologies, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Cas9/sgRNA have been developed to modify genomes of different organisms. Cas9/sgRNA has been developed from type II clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein Cas9. In Streptococcus pyogenes CRISPR system, Cas9 can be guided to specific genomic loci by a duplex consisting of mature CRISPR RNA (crRNA) and trans-activating crRNA, where the target DNA is cleaved. The CRISPR-associated Cas9 endonuclease contains the HNH and RuvC nuclease domains, which are responsible for cleavage of both strands of the target DNA.
CRISPR/Cas9 system has been simplified to a two-component system-Cas9/sgRNA, in which a synthetic single-guide RNA (sgRNA) can guide Cas9 to perform sequence-specific genome editing. Cas9/sgRNA system is attracting mounting attention since sgRNA composed of approximately 20 nucleotides is readily engineered, and multiple sgRNAs can be used to introduce mutations in several genes simultaneously, and well-designed sgRNA and Cas9 can lead to efficient mutations with minimal off-target effects. Cas9/sgRNA-mediated genome editing has been reported in bacterium, yeast, zebra fish, mice, rat, monkey, and plants. Till now, Cas9/sgRNA system has been successfully employed for genome modification in several plant species, including rice, wheat, tobacco, Arabidopsis, sorghum, tomato, maize, soybean and citrus.
In Valencia sweet orange (Citrus sinensis) and Duncan grapefruit (C. paradisi Macf.), Cas9/sgRNA system was employed to modify citrus CsPDS gene via Xcc-facilitated agroinfiltration, an optimized transient expression method in citrus. However, Cas9/sgRNA has not been harnessed for genome editing in transgenic citrus. Xcc, the causal agent of citrus canker, is a gram-negative bacterium that can infect most of citrus species, though some species are more susceptible than others. Via type III secretion system, a repertoire of Xcc-derived effectors, including transcription activator-like effector (TALE) PthA4, are injected into citrus host cells to suppress plant basal defenses, interfere with plant cellular processes to favor the pathogen growth and promote canker development. As a member of Xanthomonas AvrBs3 family-type III effectors (Boch and Bonas, 2010), PthA4 contains N-terminal translocation signal, 17.5 tandem repeat units of 34-amino-acids, three nuclear localizing signals and an acidic activation domain at its C-terminal end. Through its unique repeat units, PthA4 recognizes the corresponding promoter sequences in the host plant and activates the expression of citrus susceptibility genes that aid Xcc infection.
CsLOB1 is the disease susceptibility gene for citrus bacterial canker disease. Upon infection by Xcc, PthA4 is translocated from Xcc to host cells, where it induces CsLOB1 expression in a PthA4-dependent manner, leading to canker symptom development. PthA4 specifically binds to the effector binding elements (EBEPthA4) in the CsLOB1 promoter region (EBEPthA4-CsLOBP) to activate its gene expression. The sequence of the PthA4 effector binding elements is 5′-TATAAACCCCTTTTGCCTT-3′ (SEQ ID NO: 1), and its complementary sequence is 5′-AAGGCAAAAGGGGTTTATA-3′ (SEQ ID NO: 2).